Cancer research progress, having benefited greatly from studies using powerful model systems, strongly supports the hypothesis that accumulation of mutations leading to decreased genome stability is a critical early event in tumorigenesis. Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for avoidance of mutations and genome stability. During lagging strand DNA synthesis, multiple RNA primers and extended DNA-fragments are synthesized by DNA polymerase alpha (primase). However, this enzyme lacks proof reading function, different from the other DNA polymerases. Therefore, this initial RNA-DNA fragment (alpha-segment of the Okazaki fragment) is highly mutagenic and has to be processed by nuclease complexes. This proposal aims to define detailed molecular mechanism for the nuclease-driven "alpha-segment" processing or for Okazaki fragment maturation in yeast and mammalian cell systems. For the last funding period, we have defined the roles of three individual nucleases in the process, including S. cerevisiae RNase H(35), ScRad27 or human FEN-l, and exonucleases-1, and mutagenic consequences when these nucleases are defective. The current proposal focuses to test a central hypothesis that two mutually interactive nuclease complexes (DNA2-RPA and FEN-1-ROA1) sequentially process the alpha-segment of the Okazaki fragment. When the FEN-1 nuclease activity is inhibited by genetically built-in blocks, such as simple repeat sequences, Werner syndrome protein (WRN) and FEN-1 nuclease complex takes an alternative route to resolve instrinsic secondary structure of the displaced alpha-segment of the Okazaki fragment. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these three nucleases complexes collectively work towards alpha-segment processing in different scenarios and to relate in vitro and in vivo data using yeast and mammalian systems, including human cell lines and transgenic mice. Information made available from this systematic study will also establish a relationship between this mechanism, unique mutagenic phenotype(s), and development of genetic diseases.